Online Issues

<< All Back-issues

<< This Issue's Table of Contents

ILAR Journal V42(4) 2001
Fish Models in Biomedical Research

View/Download Brochure (PDF):
Xiphophorus

Xiphophorus Interspecies Hybrids as Genetic Models of Induced Neoplasia
Ronald B. Walter and Steven Kazianis

Ronald B. Walter, Ph.D., is Professor and Mitte Chair in Cancer Research at the Department of Chemistry and Biochemistry, Southwest Texas State University (SWTSU), San Marcos, Texas; and Steven Kazianis, Ph.D., is Assistant Professor of Research at the Department of Chemistry and Biochemistry, also at SWTSU.

Abstract

Fishes of the genus Xiphophorus (platyfishes and swordtails) are small, internally fertilizing, livebearing, and derived from freshwater habitats in Mexico, Guatemala, Belize, and Honduras. Scientists have used these fishes in cancer research studies for more than 70 yr. The genus is presently composed of 22 species that are quite divergent in their external morphology. Most cancer studies using Xiphophorus use hybrids, which can be easily produced by artificial insemination. Phenotypic traits, such as macromelanophore pigment patterns, are often drastically altered as a result of lack of gene regulation within hybrid fishes. These fish can develop large exophytic melanomas as a result of upregulated expression of these pigment patterns. Because backcross hybrid fish are susceptible to the development of melanoma and other neoplasms, they can be subjected to potentially deleterious chemical and physical agents. It is thus possible to use gene mapping and cloning methodologies to identify and characterize oncogenes and tumor suppressors implicated in spontaneous or induced neoplasia. This article reviews the history of cancer research using Xiphophorus and recent developments regarding DNA repair capabilities, mapping, and cloning of candidate genes involved in neoplastic phenotypes. The particular genetic complexity of melanoma in these fishes is analyzed and reviewed.

Key Words: CDKN2; melanoma; MNU; platyfish; swordtail; UV; Xmrk

Introduction

One of the oldest and best defined groups of established inbred strains consists of internally fertilized and livebearing platyfishes and swordtails of the genus Xiphophorus (Teleostei: Poeciliidae). These fishes are small (typically <50 mm standard length) and are derived from freshwater habitats in Mexico, Guatemala, Belize, and Honduras. Use of these fishes as a research model to study the genetic components of carcinogenesis has a history encompassing more than 70 yr. In the 1920s and early 1930s, the American biologist Myron Gordon and German scientists G. Haussler and C. Kosswig independently discovered that interspecies hybrids between strains of the southern platyfish Xiphophorus maculatus and the green swordtail Xiphophorus helleri develop melanomas spontaneously (Gordon 1931; Haussler 1928; Kosswig 1927, 1928). These neoplasms develop exclusively after interspecific crossing and originate from the extreme phenotypic enhancement of melanistic "macromelanophore" (Gordon 1927) pigment patterns derived from the southern platyfish. Pigment patterns in Xiphophorus are typically polymorphic and are composed of terminally differentiated micro- or macromelanophores and their precursors (melanoblasts and melanocytes) (Gordon 1927, 1959; Kallman and Atz 1966). Melanomas, which arise from phenotypic overexpression of macromelanophore pigment patterns and interspecific crossing, typically exhibit a disproportionate number of melanocytes that actively proliferate without sufficient regulation (Anders 1991; Gordon 1959; Schartl 1995; Vielkind 1976; Vielkind et al. 1989).

Although humans do not possess a cell type equivalent to Xiphophorus macromelanophores, human melanomas are similarly composed of improperly regulated melanocytes (Kraehn et al. 1995; Sauter and Herlyn 1998; Welch and Goldberg 1997). Additionally, in both human and Xiphophorus, melanocytes of these distantly related vertebrates are derived from the embryonic neural crest (Gordon 1959; Humm and Young 1956). Melanomas from fish and mammals also exhibit similar histopathological characteristics (Ishikawa et al. 1975; Sobel et al. 1975; Vielkind and Vielkind 1970; Vielkind et al. 1971; Yanar et al. 1996). Tumor similarity is underscored by the observation that fish melanoma cells proliferate in a manner virtually identical to those from humans after transplantion into thymus-aplastic nude mice (Schartl and Peter 1988). Fish melanoma cells are able to undergo serial passage, whereas xenografts from other nonmammalian vertebrates (reptiles and amphibians) generally exhibit rapid degeneration (Reed and Manning 1978). Melanomas from diverse vertebrates reveal a similar expression of extracellular gangliosides (Felding-Habermann et al. 1988). These studies clearly document the similarity between melanoma tumors from Xiphophorus and those from humans.

Herein, we review the results of research that has used the Xiphophorus genetic system as an experimental model to study melanoma formation and tumorigenesis in general. Although the Xiphophorus system is well suited to investigate the genetics of carcinogenesis, the attributes and variability between Xiphophorus species make this system equally valuable to investigate multifactorial genetic inheritance of any complex trait (e.g., behavior and optic or auditory sensing). Thus, we first outline below the availability of strains and describe the Xiphophorus Genetic Stock Center.

Resources for Xiphophorus Research

Dr. Gordon realized that precise identification of genes responsible for development of cancer would require genetically inbred platyfish and swordtails. Therefore in 1939, he established the Xiphophorus Genetic Stock Center and maintained it until his death in 1959. For the ensuing 30 yr, the Stock Center was maintained and expanded by Dr. Klaus Kallman at the New York Aquarium. Upon Dr. Kallman's retirement in 1993, the Xiphophorus Genetic Stock Center was transferred to Southwest Texas State University in San Marcos, Texas. Several of the original strains of platyfish and swordtails developed by Dr. Gordon in the 1930s are still available today. These strains comprise the products, in some cases, of more than 94 generations of brother-to-sister matings. This animal resource has enabled scientists to use defined genetic lines across the globe and has greatly facilitated genetic understanding of both the genus and the expansion in number and variety of interspecies tumor models. The Stock Center maintains more than 60 pedigreed Xiphophorus lines that represent 20 of the 22 species. Models of both spontaneous and induced carcinogenesis for several tumor varieties can be produced by select backcross matings between pairs of the 22 described Xiphophorus species. Examples of a few of these appear in Table 1. Information regarding Xiphophorus and availability of fish can be obtained online by accessing <www.xiphophorus.org> and using the links provided.

Xiphophorus are also actively used in other fields of study including evolution (Meyer 1997; Meyer et al. 1994), behavioral ecology (Beaugrand and Goulet 2000; Hoefler and Morris 1999; Rosenthal and Evans 1998; Trainor and Basolo, 2000), toxicology (De Wolf et al. 1993), parasitology (Dove 2000; Schmahl et al. 1996), and immunology (McConnell et al. 1998). Research within these fields also may focus on the identification of genetic factors associated with complex phenotypes. Therefore a well-developed Xiphophorus gene map, with supporting methodologies that enable quick and reliable isolation of genes, is a primary research interest within the Stock Center and collaborating research laboratories. Current gene map development is focused on the use of isozyme, restriction fragment length polymorphism, arbitrarily primed-polymerase chain reaction/randomly amplified polymorphic DNA , and microsatellite methodologies to increase marker saturation. Construction of the first complete (24 linkage groups for 24 pairs of acro- or telocentric chromosomes) genetic linkage map is currently under way (Kazianis et al. 1996, 1998b; Morizot et al. 1998a and unpublished).

Gordon-Kosswig Melanoma Model

Among the 22 recognized Xiphophorus species (Meyer et al. 1994; Rauchenberger et al. 1990), a very large number of interspecific hybrid crosses have been produced that result in phenotypic overexpression of melanistic pigment patterns derived from one of the progenitor species (Anders et al. 1973a; Gordon 1931; Kallman and Atz 1966; Kazianis et al. 1998b; Weis and Schartl 1998; Zander 1969). Examples of these crosses are presented in Table 1. However, the majority of contributions toward the understanding of Xiphophorus melanomas is derived from one specific hybrid cross (Anders 1967). This cross (Plate 1A) pairs the southern platyfish, X. maculatus, and the green swordtail, X. helleri. These two species, which very rarely develop neoplastic lesions, are sympatric over part of their range. However, they can be hybridized through artificial insemination (Clark 1950). The platyfish-specific macromelanophore pigment locus known as spotted dorsal (Sd¹) is located on the subtelomeric region of the X chromosome of X. maculatus (Ahuja et al. 1979; Nanda et al. 2000). Sd is phenotypically enhanced in first filial generation (F₁)¹ hybrids. Within the first generation backcross (BC₁)¹ to X. helleri, a Mendelian 1:1 segregation is reported between individuals with and without the Sd locus. In addition, there is a 1:1 distribution of fish revealing moderate enhancement of Sd, thus resembling the F₁ hybrid progenitors and individuals that exhibit a greatly enhanced phenotypic expression of this pigment pattern. Within this latter class of fish, exophytic nodular melanomas form, which eventually leads to necrosis of the dorsal fin and surrounding tissues. Dr. Fritz Anders (1967) attributed these phenomena predominantly to two distinct genes: the Sd locus, which resides on the X chromosome of X. maculatus, and an autosomal locus, which he termed repression gene 1 (RG₁)¹. He speculated that the Sd locus had the potential to be improperly regulated within a hybrid context, and this gene hypothetically could be regulated by the RG₁ gene. In modern terminology, these loci equate to those of an oncogene (Sd) and tumor suppressor (RG₁). The hypothetical tumor suppressor locus was later renamed Mel Sev (Morizot and Siciliano 1983; Siciliano et al. 1976) or the more common and current designation, Diff (Vielkind 1976). Genetic mapping studies subsequently localized Diff to an autosome represented by Xiphophorus linkage group (LG¹) V (Ahuja et al. 1980; Fornzler et al. 1991; Morizot and Siciliano 1983).

Histopathological studies of melanoma cells from Gordon-Kosswig tumor-bearing BC₁ hybrid fish revealed a marked decline in the differentiated state of melanin-containing cells compared with the lightly pigmented siblings (Ahuja et al. 1980; Siciliano et al. 1976; Vielkind 1976). Although the normal macromelanophore spots within X. maculatus are composed mainly of macromelanophores, melanotic tissues within hybrids reveal greater numbers of actively proliferating melanocytes, and this effect is manifested particularly within melanomas (Ahuja et al. 1980; Vielkind 1976).

The genetic model developed for Mendelian segregation of phenotypes in the Gordon-Kosswig model is believed to require two events. The first prerequisite is overexpression of a copy of a melanoma-determining gene (variously termed, Tu, erb-B*a, ONC-Xmrk, and Xmrk-2; Ahuja and Anders 1976; Wittbrodt et al. 1989; Woolcock et al. 1994; Zechel et al. 1989), which is tightly linked to the Sd pigment pattern locus on the X chromosome. For clarity herein, we use the term Xiphophorus melanoma receptor tyrosine kinase-2 (Xmrk-2)¹ to indicate this oncogene. In addition to overexpression of Xmrk-2, the second prerequisite of tumorigenesis is loss of the X. maculatus Diff tumor suppressor locus, which normally regulates growth of macromelanophores and melanocyte precursor cell populations in parental X. maculatus fish. Hypothetically, either X. helleri does not have Diff or it fails to regulate macromelanophores and their precursors properly. Thus, F₁ hybrids hypothetically harbor one functional copy of Diff, as do 50% of BC₁ hybrid animals. The F₁ hybrid fish would hypothetically exhibit moderate phenotypic enhancement of Sd. Among the BC₁ hybrids, 50% of the Sd-bearing individuals would not inherit Diff from the platyfish and thus would reveal melanosis and melanoma.

Xmrk-2 Oncogene and Xiphophorus Melanoma

Considerable work has focused on isolation and molecular characterization of candidate genes having functions ascribed to the two antagonistic melanoma regulators based on the "two-hit" Gordon-Kosswig model. Studies conducted over the past several years have resulted in the isolation and characterization of the Xmrk-2 oncogene and have demonstrated its association with melanoma in the Gordon-Kosswig hybrid cross and several other Xiphophorus tumor models. The Xmrk-2 locus, and a related proto-oncogene Xmrk-1, were cloned using reverse genetic approaches (Wittbrodt et al. 1989; Zechel et al. 1989), and they have been mapped within 0.6 cM of each other on the X. maculatus X chromosome (Gutbrod and Schartl 1999; Schartl 1990). Both loci are tightly linked to the Sd pigment pattern locus and code for transmembrane receptor tyrosine kinases, which are similar in structure to the human epidermal growth factor receptor (HER-1)¹ (Wittbrodt et al. 1989). Each of the Xmrk genes encompasses ~23 kb of genomic sequence and possesses exon/intron structures nearly identical to higher vertebrate receptor tyrosine kinases (Gutbrod and Schartl 1999). Xmrk-1 and Xmrk-2 can be distinguished by either restriction fragment length polymorphisms or polymerase chain reaction amplification using selected primers within genomic areas of divergence (Weis and Schartl 1998; Woolcock et al. 1994). The Xmrk-1 gene, which is considered the "normal" or "proto-oncogenic" gene copy, produces a 5.8-kb transcript, whereas the Xmrk-2 "oncogenic" copy produces a shorter, 4.7-kb transcript (Adam et al. 1991). The Xmrk-1 transcript is expressed at low levels in all tissues tested (using Northern blot hybridizations; Dimitrijevic et al. 1998; Wittbrodt et al. 1989) and is developmentally regulated in embryos inasmuch as the level of RNA expression is high in the early stages (0-4; also found in unfertilized eggs), declines during cleavage and neurula stages, and is expressed once again during organogenesis (Wittbrodt et al. 1989). In contrast, the Xmrk-2 4.7-kb transcript is virtually undetectable in normal tissues using Northern blot methodologies and is distinctly overexpressed in melanomas derived from F₁ and BC₁ hybrids within the Gordon-Kosswig cross. These results have been collaborated by additional studies using reverse transcription polymerase chain reactions and Western blotting (Dimitrijevic et al. 1998; Wellbrock et al. 1998a; Woolcock et al. 1994).

Xmrk-2 encodes a 160-kDa protein. The predicted secondary structure consists of a cytoplasmic tyrosine kinase subunit connected by two cysteine-rich hydrophobic transmembrane sequences to an extracellular ligand-binding domain (Wittbrodt et al. 1989). Analysis of several X. maculatus mutant fish lines (arising spontaneously or through x-ray irradiation) revealed insertions or deletions within the Xmrk-2 oncogene, and BC₁ hybrids that had been produced by crossing these mutants with X. helleri failed to show melanotic hyperplasia/tumorigenesis (Schartl et al. 1999; Wittbrodt et al. 1989). When Winkler and colleagues (1989) placed the Xmrk-2 coding region under strong constitutive promoter regulation and used it to microinject medaka (Oryzias latipes) embryos, several kinds of tumors developed very early, in some cases resulting in embryonic lethality (Winkler et al. 1994).

At the intracellular level, gene fusion experiments between the Xmrk-2 intracellular domain and the extracellular ligand binding domain of the human HER-1 protein have shown that Xmrk-2 is able to bind or phosphorylate a number of intracellular targets, including the ubiquitous general receptor tyrosine kinase substrate phospholipase C-gamma and cytoplasmic kinases src, fyn, yes, Shc, and the adaptor protein GRB2 (Wellbrock et al. 1995, 1998a, 1999). Constitutive expression of Xmrk-2 also leads to activation of a transcriptional regulator STAT5 and subsequent upregulation of downstream target loci (Wellbrock et al. 1998b). In addition, Wellbrock and Schartl (1999) have shown that phosphotidylinositol-3 kinase (PI3 kinase¹) can associate with the intracellular portion of Xmrk-2 and become activated via this interaction. PI3 kinase is known to be responsive to growth factors and to play an intermediate role in cell cycle progression. Whether the PI3 kinase alone, or one or more Xmrk-2-associated factors are the true mediators of downstream signal transduction contributing to the Gordon-Kosswig melanoma phenotype is still under investigation. Clearly the recent emphasis on biochemical studies to identify intracellular proteins targeted by the Xmrk-2 kinase are well under way and should illuminate our understanding of this interesting model in the near future.

In an effort to identify potentially important differences between Xmrk-1 and Xmrk-2, researchers have determined the cDNA and genomic sequences of both genes (Adam et al. 1991; Gutbrod and Schartl 1999). Of note are several genomic sequence differences between them, including two deletions (1344 and 581 bp) found exclusively in Xmrk-2 alleles. The first deletion is an in-frame deletion located only in one Xmrk-2 allele, and the second deletion is in the last exon of two studied alleles, downstream from polyadenylation consensus sequences (Adam et al. 1991). These changes are not considered to be associated significantly with the tumorigenicity of Xmrk-2 (Adam et al. 1991; Gomez et al. 2000). However, analysis of the coding region at the 5' end of these genes has recently revealed two key codon differences between them (Dimitrijevic et al. 1998; Gomez et al. 2000). These differences are within the extracellular domain (C578S and G359R), resulting in ligand-independent dimerization and activation of the Xmrk-2 protein (Dimitrijevic et al. 1998; Gomez et al. 2000). Furthermore, the ability of these differences to lead to tumor development has been tested by in vivo experiments that involve microinjection of medaka embryos and result in the different tumorigenic potency of Xmrk-1 and Xmrk-2 genes (Winkler et al., 1994).

Although these differences may affect Xmrk (-1 and -2) protein function, they do not account for the large transcriptional differences observed between them. Extensive characterization of the promoter regions of both genes revealed significant sequence differences in regulatory elements (Adam et al. 1993; Altschmied et al. 1995, 1997; Fornzler et al. 1996; Woolcock et al. 1994). The Xmrk-1 promoter contains regulatory elements consistent with its ubiquitous and low expression pattern, and it likely provides "housekeeping" function. In contrast, the Xmrk-2 promoter region contains structural elements inconsistent with "housekeeping" genes and completely dissimilar to that of Xmrk-1. The promoter sequence of Xmrk-2 exhibits distinct DNA sequence similarity to promoters derived from a sex-linked locus referred to as Donor (or "D") locus. This gene is found in multiple copies throughout the genome of Xiphophorus and related taxa (Fornzler et al. 1996; Nanda et al. 1996). Each of the Donor loci may code for one of two distinct polypeptides. One of these genes exhibits a high level of homology to a zinc finger protein of the krüppel type (Schuh et al. 1986), and the other is an unknown gene with great similarity to a putative gene sequence from Caenorhabditis elegans (F54H12.3; Fornzler et al. 1996). Researchers have proposed that the divergent promoters of the two Xmrk genes have arisen from an ancient gene duplication, presumably involving a nonhomologous recombination event (Adam et al. 1993; Fornzler et al. 1996).

Detailed study of the Xmrk-2 promoter resulted in the identification of a critical GC-box element, which sequesters proteins with structural similarity to mammalian Sp1 transcription factors (Baudler et al. 1997). Also, the Xmrk-2 promoter region contains a 5' CpG island. Cytosines within this genomic region are methylated in tissues derived from nonhybrid fish, but they are unmethylated in melanotic tissues derived from hybrids and in a melanoma-derived cell line (Altschmied et al. 1997). Thus, such differential methylation might contribute to Xmrk-2 expression and the development of melanomas (Altschmied et al. 1997).

In summary, it is evident that Xmrk-2 differs from the Xmrk-1 proto-oncogene in two significant ways: (1) through a mutation in the extracellular protein domain, which leads to a potent, constitutive tyrosine kinase receptor activity that exhibits ligand-independent activation, and (2) via constitutive oncogenic RNA/protein overexpression in melanotic tissues derived from hybrid fish. However, because the parental X. maculatus Jp 163 A stock very rarely develops melanoma, these phenomena alone clearly do not independently lead to spontaneous melanoma within Gordon-Kosswig BC₁ hybrids. Melanoma formation is also mediated by the Diff locus, located in Xiphophorus LG V. Consequently, much recent effort has focused on gene mapping and candidate gene isolation of the hypothetical Diff tumor suppressor gene.

Diff Tumor Suppressor

Evidence that Diff could be a prominent locus involved in melanoma formation within the Gordon-Kosswig cross was assembled from linkage analyses to polymorphic isozyme markers analyzed in BC₁ hybrids (Ahuja et al. 1980; Fornzler et al. 1991; Morizot and Siciliano 1983; Siciliano et al. 1976). Until recently, no reasonable candidates for Diff could be proposed. Researchers have mapped known vertebrate tumor suppressor (TP53; Kazianis et al. 1998a; Nairn et al. 1996a), oncogene (JUNA, JUNB, ERBB, SRC, Xyes, and Xfyn; Hannig et al. 1991; Morizot et al. 1998a and unpublished), and DNA repair genes (ERCC2/XPD, and LIG 1; Della Coletta et al. 1995, Walter et al. 1993) orthologs in Xiphophorus to other linkage groups and have failed to evidence genetic association with melanoma formation in BC₁ hybrids. More recent development of modern gene mapping methodologies has enabled the production of a finer scale map and consequently finer localization of the Diff tumor suppressor gene on LG V (Kazianis et al. 1998b, 1996; Morizot et al. 1998b).

Recently, scientists cloned a fish homolog of the cyclin-dependent kinase inhibitor-2 (CDKN2, also known as INK4) gene family from the Xiphophorus genome (Kazianis et al. 1999; Nairn et al. 1996b). This gene, designated CDNK2X (see below), was subsequently mapped to LG V and has become the primary candidate for Diff. The CDKN2 family of proteins is implicated in the regulation of the G1 checkpoint phase of the cell cycle and modulate the kinase activity of CDK4 and CDK6, which in turn regulate the phosphorylation/inactivation of the Rb tumor suppressor. Inactive Rb leads to the release of several proteins, including members of the E2F transcription factor family, which directly regulate the expression of genes involved in S phase (Chin et al. 1998; Drexler 1998; Ruas and Peters 1998). The coding sequence of CDKN2X reveals significant homology to but is equally distant from mammalian CDKN2A (P16) and CDKN2B (P15) tumor suppressor genes (Kazianis et al. 1999). Because primary structure alignments with all four known mammalian gene family members made assignment of orthology impossible, the gene was designated CDKN2X (wherein the "X" after the gene designation denotes the Xiphophorus form; Kazianis et al. 1999). The derived CDKN2X polypeptide codes for a 13-kDa protein, a size that has been confirmed by Western blotting (M. Schartl, University of Würzburg, personal communication, 2000). All known CDKN2 gene family members share a protein structure composed of four to five ankyrin repeats (Chin et al. 1998; Drexler 1998; Luh et al. 1997; Ruas and Peters 1998; Venkataramani et al. 1998), and the fish polypeptide p13^CDKN2X possesses four of these, similar to mammalian p15^CDKN2B (Kazianis et al. 1999). CDKN2X has been sequenced from both X. maculatus and X. helleri. The two species exhibit only two conservative amino acid differences, neither of which would be expected to alter function (Kazianis et al. 1999). In both species, as in the human CDKN2B locus, there is a single intron separating two exons (Jiang et al. 1995).

The map position of the CDKN2X gene correlates with localization of Diff in Xiphophorus LG V (Kazianis et al. 1998b). Analysis of melanoma-bearing BC₁ hybrids revealed that the vast majority (136/165) of these fish did not inherit the X. maculatus CDKN2X allele (Kazianis et al. 1998b). A similar relation was evident between phenotypic severity of pigment pattern enhancement and CDKN2X allelotype in that heavily pigmented BC₁ were usually homozygotes for the X. helleri alleles whereas lightly pigmented fish retained one X. maculatus CDKN2X allele (see Plate 1A and Table 2). Additionally, fine mapping of the CDKN2X locus with other flanking LG V markers, and analysis with pigmentation degree as a quantitative trait locus, indicated a peak of association between pigmentation and LG V markers that centered on the CDKN2X locus (likelihood ratio of >10; Kazianis et al. 1998b). Based on these cumulative results in addition to the documented involvement of mammalian CDKN2 gene family homolog P16 in human melanoma (Chin et al. 1998; Drexler 1998; Ruas and Peters 1998), CDKN2X is a candidate for the historical Diff tumor suppressor gene proposed in the Gordon-Kosswig classical melanoma model (Kazianis et al. 1998b, 1999, 2000).

To assess potential involvement of CDKN2X in fish melanomagenesis, scientists have determined the genomic structure and expressional characteristics in parental fish (X. maculatus and X. helleri) used in the Gordon-Kosswig cross. One of the CDKN2X promoter region's two CpG islands is located immediately upstream of the start of transcription and continuing into exon 1, and the other is within the 5' end of exon 2. This organization is evolutionarily conserved in other CDKN2 gene family members (reviewed by Kazianis et al. 2000). After promoter hypermethylation leading to tumor suppressor gene silencing had been documented for mammalian CDKN2A and CDKN2B (Herman et al. 1995, 1996; Malumbres et al. 1999), researchers sought to assess the methylation status of the CDKN2X locus. Both of the CpG islands proved to be unmethylated at virtually every CpG dinucleotide site (Kazianis et al. 2000 and unpublished). In stark contrast, our analysis of 10 other Xiphophorus genes, including DNA repair genes (uracil-N-glycosylase, ERCC2/XPD, and DNA ligase 1), tumor suppressors and oncogenes (TP53, JUNA, and Xmrk-1), and general function genes (ß-actin, JUNB, 5-methylcytosine methyltransferase, S15-rig, and Xmrk-1), indicates that they are methylated at every available CpG site in all tissue sources examined, despite the presence of CpG islands equal to those in CDKN2X with regard to GC content and length (Walter and Li unpublished). However, analyses of CDKN2X methylation in many different tissues from F₁, BC₁ hybrids, and parental controls failed to reveal significant differences in CDKN2X promoter region methylation. Therefore, it is unlikely that methylation plays a prominent role in CDKN2X gene silencing and melanoma tumor suppression (Kazianis et al. 2000).

The CDKN2X promoter region exhibits distinct differences between X. helleri and X. maculatus, including a 20-bp region that is absent in X. maculatus and, conversely, an expanded GT-repeat sequence (80 GT-repeats) present in the southern platyfish but much reduced (11 GT-repeats) in the green swordtail (Kazianis et al. 1999). In several BC₁ hybrid tissues, the X. maculatus CDKN2X allele is expressed at higher levels than the X. helleri allele, and this effect is manifested particularly in melanotic tissues (Kazianis et al. 1998b). Reduced expression of the X. helleri allele in melanotic tissues would be a predicted result if CDKN2X were functioning as a tumor suppressor gene. Thus, the experimentally established differential CDKN2X expression patterning is consistent with its putative function as Diff. In addition, reduced CDKN2A (P16) expression in humans, which occurs via several distinct mechanisms, has been implicated in melanoma (Chin et al. 1998; Drexler 1998; Ruas and Peters 1998). However, detailed examination revealed that RNA expression of CDKN2X was more robust in numerous Xiphophorus melanomas such as those located on skin and fin than in control tissues (Kazianis et al. 1998b, 2000). These results have led to an alternative hypothetical model in which CDKN2X expression levels in melanocytes may influence melanocyte differentiation in hybrid fish. If the CDKN2X gene is inadequately expressed in BC₁ hybrids lacking X. maculatus alleles, melanocytes might not fully differentiate into macromelanophores; and this lack of complete differentiation, coupled with Xmrk-2 oncogene activation, could lead to a path of tumorigenesis. Such a scenario is paralleled by results derived from laboratory mouse studies, wherein overexpression of an activated H-RAS (G^12V) transgene in a CDKN2A null background resulted in a very high incidence of melanoma (60% at 6 mo of age; Chin et al. 1997). Furthermore, the same transgene in a hemizygous CDKN2A genetic background resulted in a low tumor incidence, and genetic analysis of the rare tumors that did occur revealed the presence of homozygous CDKN2A deletions (Chin et al. 1997). RAS proteins are activated within the cytoplasmic tyrosine kinase cascade by several proteins, including GRB2 and Shc, and these in turn are activated by receptor tyrosine kinase proteins (Chin et al. 1998). Because Xmrk-2 has been shown to bind/activate both GRB2 and Shc (Wellbrock and Schartl 1999), it is not difficult to envision a parallel between the RAS/CDKN2A mouse model and a RAS-mediated antagonistic relation between Xmrk-2 and CDKN2X within Xiphophorus fish. Obviously, numerous other hypotheses of involvement exist for both the Xmrk-2 and CDKN2X genes that remain to be investigated, including potential use of transgenic or "knock-out" Xiphophorus, as the technologies become available (Chen et al. 2001). Further study of chromosomal regions and loci surrounding these genes is currently at the forefront of studies (Nanda et al. 2000; Volff et al. 2001 and unpublished) and will hopefully elucidate the precise molecular mechanisms of melanoma formation in these fishes.

Relation of Inducible Xiphophorus Tumor Models to the Gordon-Kosswig Model

The foregoing discussion highlights progress toward understanding the molecular basis of the two-gene hypothesis for segregation of melanoma in the Gordon-Kosswig model. However, researchers have also documented several tumor types that are not associated with inheritance of CDKN2X (or LG V) among the variety of Xiphophorus hybrid model crosses. Many of these model crosses require pretreatment of BC₁ animals with DNA-damaging agents (ultraviolet light [UV¹] or N-methyl-N-nitrosourea [MNU¹]) soon after birth to express tumor development. Depending on the cross and the inducing agent used, treated BC₁ hybrids exhibit from 2 to 30% tumor incidence at 6 to 8 mo of age (see below). Investigation of the genetics underlying these inducible Xiphophorus tumor models is a major focus of current Xiphophorus research.

The variability of pigment patterns and the modification of their expression after selective interspecies hybridization among Xiphophorus species present many interesting models for examining induced tumorigenesis of cells derived from the neural crest. An example of differences in pigment pattern expression on interspecies hybridization is shown in Plate 2. Because they have been derived from a single female, the X. maculatus lines Jp 163 A and Jp 163 B are very closely related; however, they are separated and maintained as inbred lines (94 and 89 generations, respectively) based on their distinct macromelanophore pigment patterns. When X. maculatus harboring the spotted dorsal (Sd¹; strain Jp 163 A) or spotted side (Sp¹; strain Jp 163 B) macromelanophore pigment pattern are hybridized with another platyfish Xiphophorus couchianus (inbred 63 generations), the Sp pattern becomes enhanced in Sp-inheriting BC₁ hybrids and resembles the melanotic enhancement observed for Sd in the Gordon-Kosswig cross (Plates 1 and 2). However, when X. maculatus carrying the Sd pattern (i.e., Jp 163 A) are crossed with X. couchianus, the Sd character becomes largely suppressed (Plate 2A). This example illustrates that among Xiphophorus crosses, variability of pigment pattern expression in BC₁ hybrids can be experimentally manipulated to test comparative hypotheses regarding the nature of gene association in a wide array of tumor types (see Table 1).

UV-induced models that exhibit inheritance patterns similar to the Gordon-Kosswig melanoma cross are described below. Chemical (MNU) induction of tumors in the BC₁ hybrids, which appears to involve genetic mechanisms that may be quite different from UVB-induced tumorigenesis, is considered in the subsequent section.

Xiphophorus UV-induced Melanoma Models

Much of the historical work on tumor development in Xiphophorus pertains to the Gordon-Kosswig melanoma model wherein melanomas develop spontaneously in a predictable proportion of BC₁ hybrids due to the allelic segregation at two specific loci in the particular interspecific cross (Anders 1991; Schartl 1995). More recently, additional Xiphophorus backcross hybrid models have been developed wherein BC₁ animals normally develop tumors at a very low incidence (~7%) but in which high incidence of melanoma can be induced readily by subjecting young fish (5 days after birth) to UV (Nairn et al. 1996c; Setlow and Woodhead 1994; Setlow et al. 1989, 1993). Researchers have used these UV-inducible tumor models to help define specific wavelengths that are melanomagenic and for use as comparative genetic models vis-à-vis the Gordon-Kosswig cross (Nairn et al. 1996c; Setlow and Woodhead 1994; Setlow et al. 1989, 1993).

Although controversial, it is believed that cutaneous malignant melanoma in humans often develops when genetically predisposed individuals are exposed to environmental agents, such as excessive sunlight. Cutaneous malignant melanoma incidence is greatly elevated, and onset is accelerated in certain families in which genetic predisposition is obvious, although these cases of strong heritability may account for only ~10% of malignancies (Goldstein et al. 1994). Nonheritable melanoma is an important public health concern because of an alarming recent increase in worldwide incidence, perhaps attributable to depletion of stratospheric ozone and the widespread practice of cosmetic skin tanning (deGruiji and Van der Leun 1993; Koh 1991; Rigel et al. 1987). From 1973 to 1990, the incidence of cutaneous malignant melanoma in the United States increased ~94%---more than that for any other cancer (Miller et al. 1993).

Although the maximal absorption for nucleic acids is in the UVC wavelengths (230-290 nm), UVB (290-320 nm) as well as UVA (320-400 nm) radiation may also alter genetic information and leave characteristic mutational "signatures" in altered genes (Drobetsky et al. 1995; Pollock et al. 1996; Wikonkal and Brash 1999). However, the relation between sunlight exposure and melanoma incidence is complex and may depend on the quantity and temporal specifics of exposure (de Gruijl 1999; Langley and Sober 1997). Hereditary factors also contribute to melanoma development in humans (Kamb et al. 1994; Langley and Sober 1997; Laporte 1998), and hypotheses attempting to correlate the dynamics of sunlight exposure and melanoma incidence must also include consideration of the genetic determinants underlying melanoma incidence. Although this caveat is generally recognized, it underscores the need for animal models for melanoma in which genetic components are easily recognized and subject to experimental manipulation and analysis.

The first UV-inducible Xiphophorus tumor models used BC₁ hybrid fishes produced from mating X. maculatus Jp 163 B carrying the spotted side (Sp) pigment pattern with the swordtail X. helleri (Setlow et al. 1989) (Plate 1B). This cross is very similar to the Gordon-Kosswig cross but differs in that the BC₁ hybrids do not develop tumors at appreciable incidences without UV treatment very soon after birth (Table 3). These BC₁ hybrid fish proved to be susceptible to development of neoplastic disease, which was inducible by exposure to wavelengths in the UVB range (Setlow et al. 1989). In addition, the melanoma incidence could be reduced by exposure to visible light immediately after the UVB treatment, indicating that photoreversal of UV-induced DNA damage played a role in the ultimate effects of UVB-induced carcinogenesis (Setlow et al. 1989). Later experiments using Xiphophorus BC₁ hybrid fish models and discrete wavelengths of UVA and visible light (405 and 436 nm) indicated that long wavelength light could also induce melanoma. This observation has considerable importance considering the composition of many sunscreens (Setlow and Woodhead 1994; Setlow et al. 1993). Because the majority of UV radiation reaching the earth surface is within the UVA range (Setlow 1974; Setlow et al. 1993), these experimental results prompted concern over the epidemiology of melanoma in humans and the use of certain sunscreens designed to minimize erythema induced by UVB radiation.

Genetic analyses of UVB-induced melanoma using the X. helleri ´ (X. maculatus Jp 163 B ´ X. helleri) (Plate 1B) model cross indicates that inheritance of the Xmrk and CDKN2X genes is strongly associated with BC₁ hybrid melanomagenesis (Table 2; Kazianis et al. 1998a,b; Nairn et al. 1996c). Similar to the Gordon-Kosswig model, all of the animals that develop melanoma inherit an Xmrk-2 allele from the X. maculatus parent. In addition, approximately 80% of UVB-exposed BC₁ hybrid animals that develop melanoma inherit both CDKN2X alleles from the X. helleri parent. Furthermore, the degree of pigmentation (lightly vs. heavily pigmented, see legends in Figure 1 and 2) in BC₁ hybrids is also strongly associated with CDKN2X inheritance (Table 2). However, the fact that these animals develop melanoma more frequently than the nonirradiated controls indicates that other genes must also be segregating into the certain BC₁ hybrids, predisposing a portion of them to the effects of UV radiation. In addition, it is important to note that association with CDKN2X inheritance in melanoma-bearing UVB-treated BC₁ hybrids is not complete. Mechanisms accounting for the ~20% of melanoma-bearing BC₁ fish that are genotypically heterozygous for CDKN2X may involve loss of function of the X. maculatus allele due to UV-induced mutagenesis or UV-induced regulatory modulation. These and other possibilities are currently under investigation.

Genetic analyses of the above-mentioned hybrid model and others indicate independent molecular genetic smechanisms leading to melanomas induced by UV wavelengths, as opposed to those arising spontaneously (or induced by 405 nm of light; Nairn et al. 1996c). The use and future exploitation of these and other Xiphophorus melanoma models for UV carcinogenesis is an exciting prospect, both for determination of the melanoma action spectrum and for continued genetic analysis of the hereditary factors involved in UV-induced melanoma formation and progression.

Chemical Carcinogen-induced Models

Less well developed than the spontaneous and UV-induced Xiphophorus tumor models, but no less important, are induced tumor models that use chemical (e.g., MNU) treatment as a tumor-inducing stimulus (Kazianis et al. 2001a; Schwab et al. 1978a,b, 1979). MNU is an alkylating agent that methylates DNA bases primarily at nucleophilic sites (N⁷ and N³ alkylpurines). The primary mutagenic lesion of MNU exposure is believed to be O⁶ methylguanine (Friedberg et al. 1995). MNU induces numerous cancers in rodents, including mammary carcinomas and thyroid tumors in rats (Ohshima and Ward 1984; Zarbl et al. 1985) as well as thymic lymphomas in mice (Frei and Lawley 1980; Richie et al. 1996). This carcinogen also has been shown to induce a wide variety of tumors in Xiphophorus hybrids, including neuroblastomas, melanomas, fibrosarcomas, rhabdomyosarcomas at high incidence, and various carcinomas at a greatly reduced incidence (Schwab et al. 1978a,b, 1979).

Schwab and colleagues (1978a) were the first to indicate that chemically induced tumorigenesis in Xiphophorus possesses a strong genetic component. Scientists have shown that MNU treatment of 64 independent nonhybrid species/strains and derived hybrids induces neuroblastomas exclusively in BC₁ animals derived from one particular hybrid cross involving Xiphophorus variatus and carrying the Lineatus (Li) pigment pattern hybridized with X. helleri (Schwab et al. 1978a,b, 1979). However, these studies have not been developed past the initial description of tumor types and treatments.

We have recently developed several new MNU-inducible hybrid models that promise to further advance Xiphophorus as an important model for the study of chemical carcinogenesis (Kazianis et al. 2001a,b and unpublished). To provide a direct comparison between UVB and MNU tumor induction, we exposed X. helleri ´ (X. maculatus Jp 163 B ´ X. helleri) (Plate 1B) BC₁ hybrids to MNU. As a comparative example, the data in Tables 3 and 4 illustrate that the level of MNU induction of melanoma is higher than by UVR, and that there is no significant association with CDKN2X inheritance in BC₁ melanoma-bearing animals. This absence of association with CDKN2X suggests that the inheritance of different gene targets may be necessary to predispose BC₁ animals to UV or MNU tumor induction. Also, among the MNU-treated melanoma-bearing animals, approximately 20% exhibit multiple neoplastic lesions, often in distinct body areas, a situation that is not observed among hundreds of UVB-induced tumor-bearing fish. The UVB and MNU results comparatively indicate that multiple genetic routes may exist that lead to the same tumor (i.e., melanoma) even within BC₁ hybrids produced from pedigreed and highly inbred parental lines.

In a different model cross, Xiphophorus andersi ´ (X. maculatus Jp 163 B ´ X. andersi), the BC₁ exhibit enhancement of the spotted side pigment pattern nearly identical to the enhancement observed for the heavily pigmented hybrids shown in the X. couchianus backcross (Plates 2B and 3). Attempts at UVB induction of melanoma in such pigmented BC₁ hybrids have not been successful despite use of radiation protocols leading to substantial induction of melanoma in other model crosses (Nairn et al. 2001). However, MNU-induced tumorigenesis in the same hybrid model, X. andersi ´ (X. maculatus Jp 163 B ´ X. andersi), occurs at very high incidences (approaching 30% in BC₁ hybrids; Table 5). As is the case for the X. helleri ´ (X. maculatus Jp 163 B ´ X. helleri) model cross (Plate 1B and Table 4), genetic analyses did not show any association of melanoma development with inheritance of CDKN2X alleles (Table 5; Walter and Kazianis unpublished), which not only underscores the genetic complexity of melanoma but also attests to a major strength of using the Xiphophorus genetic system to identify the genetic mechanisms leading to tumor predisposition. If CDKN2X inheritance is not playing a role in MNU-induced tumorigenesis in the same model crosses where it is clearly associated with UVB-induced melanoma, what gene target(s) might be serving to predispose these hybrids to tumor development? Efforts are currently under way to perform extensive genotype analyses of MNU-treated BC₁ hybrids from the above-mentioned cross involving X. andersi. The robust genetics offered by interspecies hybridization will hopefully allow identification of factors associated with MNU tumor induction that will be of comparative interest.

A strictly MNU-induced tumor model that has recently been developed involves backcross hybrids using X. maculatus Sd (Jp 163 A) and the platyfish X. couchianus as the recurrent parent. As previously stated, when X. maculatus carrying the Sd pigment pattern are crossed to X. couchianus, the Sd macromelanophore pigment pattern becomes largely suppressed, whereas an erythrophore pattern referred to as dorsal red (Anders et al. 1973b; Kallman, 1975) becomes enhanced in BC₁ hybrids (Plate 2A). To determine whether BC₁ animals that do not exhibit melanophore enhancement might nevertheless be predisposed to induced tumorigenesis, we exposed BC₁ animals from the X. couchianus ´ (X. maculatus Jp 163 A (Sd) ´ X. couchianus) backcross to MNU. In Table 6, a data set obtained from these experiments is shown.

MNU treatment of hybrid fish derived from crossing these two platyfish species resulted in the induction of several and varied neoplasms occurring at incidences between 2.8 and 6.6%. Even though the Sd pattern is suppressed in these BC₁ hybrids, a second melanophore pattern termed anal fin spot (Af¹) is expressed as a very small area of melanocytes located at the distal tip of the gonopodium (Plate 4A). Surprisingly, 10.4% of MNU-treated backcross hybrids exhibited extensive melanosis emanating from the melanocytes sequestered at the tip of the gonopodium. These melanocytes gradually expanded along the gonopodium and into the ventral viscera (Plate 4B). This enhanced melanosis occurred only in MNU-treated animals (0 instances observed in equal numbers of controls). This study identified three genetic preconditions for expression of Af melanosis in BC₁ hybrids: (1) Individuals must inherit an X chromosome derived from X. maculatus that carries the Af pigment pattern locus. (2) Affected fish are always males, inasmuch as the Af pigment pattern is sex limited and only male fish develop a gonopodium and have a melanocyte population restricted to its distal end. (3) As in the UVB-induced melanomas and the Gordon-Kosswig melanoma model, but unlike MNU-induced melanomas (see above) in other crosses, the progressive Af melanosis is significantly associated with the absence of inheritance of X. maculatus CDKN2 alleles (Kazianis et al. 2001b). In this case, 100% of BC₁ fish developing Af melanosis were homozygotes for X. couchianus CDKN2X alleles. Conversely, the majority of individuals that did not develop severe Af melanosis, even after MNU treatment, were heterozygous for CDKN2X.

In addition to Af melanosis, discovery of schwannomas, fibrosarcomas, and retinoblastomas in the BC₁ hybrids, albeit at a lower incidence, creates the additional possibility of studying the genetic background underlying these neoplasms as well. We are currently initiating crosses to explore the genetic basis of these low-incidence tumors using hybrids between X. maculatus and X. couchianus and in several other crosses.

The results reviewed herein and other unpublished studies clearly indicate that MNU-induced melanomas can arise through genetic mechanisms distinct from those identified for UVB-induced tumorigensis, even within a specific Xiphophorus model crossing scheme in which both parental lines are extensively inbred. It is our hope that continued study of these various models will allow identification and isolation of genes implicated in MNU-induced melanomas and other cancers.

Spontaneous Melanoma Models in the Genus Xiphophorus

The involvement of LG V with melanoma susceptibility has been confirmed in several Xiphophorus hybrid crosses related to the Gordon-Kosswig cross (Ahuja et al. 1980; Kazianis et al. 1998b, 2001b; Morizot and Siciliano 1983). In all cases, these crosses involve X. maculatus from the Rio Jamapa, outcrossed to either X. helleri or X. couchianus. In these crosses, the involvement of a gene or genes in LG V as regulator(s) of melanotic hyperplasia is not restricted to fish with the Sd pigment pattern, but also occurs in those with Sp and Af, two distinct pigment patterns also located on the X chromosome of X. maculatus.

Genetic linkage analyses have also been conducted for several other Xiphophorus hybrid crosses. In several of these, BC₁ hybrids have exhibited extreme macromelanophore pigment pattern enhancement and melanoma formation that cannot be attributed to CDKN2X or LG V. Some of these crosses are summarized in Table 1. In one such example, X. variatus with a macromelanophore pattern referred to as Pu² is crossed to X. helleri. Although F₁ and subsequent BC₁ hybrids (to X. helleri) exhibit pigmentary hyperplasia and melanoma development, genetic analysis failed to implicate a Diff locus, as determined by analysis of several LG V markers (Kazianis et al. 1996). In contrast, researchers attributed phenotypic differences in Pu² expression in BC₁ hybrids to genotypic differences at a locus at the telomeric region of LG III (Kazianis et al. 1996; Morizot et al. 1998b and unpublished).

In addition to melanoma models induced by hybrid crossing, Xiphophorus fish also provide several relatively unexploited models of melanoma formation that do not involve interspecies hybridization (Borowsky 1973; Kallman 1971; Schartl et al. 1995). For example, X. cortezi fish with the Sc macromelanophore pattern can develop melanomas that are associated with aging (Kallman 1971; Schartl et al. 1995). The development of melanosis and melanoma also appears to be influenced by androgens, inasmuch as dominant males appear to develop the most extreme manifestations of melanosis and melanoma (Schartl et al. 1995). At the time of this writing, this model and several others that are associated with aging in Xiphophorus have not been studied extensively or with the benefit of modern applied research techniques and technologies.

Tumor Inducibility and DNA Repair Potential

The fact that different responses are observed using the same interspecies cross model that appears dependent on UV or MNU tumor induction protocols suggests that the action of these two agents may be tolerated by a given species or hybrid differentially. The parental species used in most tumor cross models are highly inbred fish lines. Thus, within feral populations of these species, a standard distribution of DNA repair phenotypes (i.e., capabilities) may exist; however, we may have unknowingly selected and genetically fixed them by inbreeding. Thus, different molecular elements and genes used in combating DNA damage within a given species may not be totally compatible with another species when placed in F₁ or BC₁ genetic backgrounds, which could result in modulated ability to clear DNA damage and a potentially tumorigenic genotype. To investigate this possibility, researchers have initiated DNA repair assays in an effort to determine the DNA repair capabilities of parental species/strains used in the tumor model crosses, as described below.

In a recently reported experiment, the swordtail Xiphophorus signum was exposed to UVB radiation. DNA from the skin and fin was then assayed at various times after exposure using radioimmunoassays specific for the major UV-induced DNA photoproducts to determine the extents of nucleotide excision repair (Meador et al. 2000). The results indicated that induction of both the cyclobutane pyrimidine dimers (CPDs¹) and 6-4 photoproducts ([(6-4)PDs¹]) in addition to the calculated half-life (initial rate of repair) of CPD and (6-4)PD nucleotide excision repair or dark-repair exhibited values very comparable with those reported for rodents (Meador et al. 2000).

Investigation of nucleotide excision repair among Xiphophorus species using the UV photoproduct radioimmunoassay (Mitchell 1996; Mitchell et al. 1993) revealed highly variable repair capabilities (Mitchell et al. 2001 and unpublished). For example, researchers have determined the kinetics for CPD and (6-4)PD repair for several species, including X. couchianus, X. maculatus (strains Jp 163 A and Jp 163 B), X. signum, X. variatus, and X. andersi. They found the relative capacity to repair CPDs and (6-4)PDs to be comparable in X. variatus, X. signum, and X. couchianus; however, these species revealed reduced repair levels compared with X. maculatus and X. andersi. The parental lines, X. maculatus Jp 163 A and X. andersi, displayed very efficient repair of CPDs and (6-4)PDs with comparable rates of removal. These data are consistent with the reported lack of UVB-induced tumorigenicity observed in hybrid fish from X. andersi crosses (Plate 3 and Table 5).

Unlike UV exposure, treating cells with monofunctional alkylating agents (e.g., MNU) induces DNA damage that is principally processed by base excision repair (BER¹). One of us with colleagues (Walter et al. 2001a,b) have used two distinct oligonucleotide-based assays---the first for uracil-N-glycosylase-initiated base excision repair and the second specifically for repair by O⁶methylguanine-DNA-methyltransferase (O⁶-MGMT¹)---to begin assessing DNA repair capability among Xiphophorus parental fish lines exposed to alkylating agents.

Both BER and O⁶-methylguanine-DNA-methyltransferase (O⁶MGMT¹) assays in Xiphophorus fishes and F₁ interspecies hybrids have indicated that various tissues exhibit different levels of repair capability. For example, brain extracts generally exhibited greater BER and O⁶MGMT repair activity than gill and liver extracts. Although we did not observe differences between species in the ability of a given tissue to repair the O⁶-methylguanine DNA lesion (O⁶-MGMT repair), we did observe species-specific differences in BER capabilities.

Comparing BER activities between each of two parental lines and their F₁ hybrids indicated that X. couchianus possessed less BER repair capability in gill and liver extracts than X. maculatus Jp 163 A. Furthermore, the repair capacity in F₁ hybrids produced by mating X. maculatus Jp 163 A with X. couchianus is very similar to values obtained for the X. couchianus parent, including a decreased ability to perform BER (compared with X. maculatus) in both gill and liver tissues. In addition, these F₁ hybrids may repair less well in gill tissue than either parent species, suggesting that the hybrid genetic background (heterozygotic for all loci) produces a genetic condition whereby protein interactions leading to efficient BER are compromised in hybrid gill tissue. In MNU tumor-induction exposures, we have observed that X. maculatus Jp 163 A ´ X. couchianus F₁ hybrids are more sensitive to MNU toxicity than either parent species (Walter unpublished).

The DNA repair studies conducted to date have not assessed repair capability in individual BC₁ hybrid animals that do or do not develop induced tumors. However, scaling down these DNA repair assays so that DNA repair capability can be treated as a quantitative trait may provide valuable data to document the relations between DNA repair and tumorigenicity. The Xiphophorus tumor models may then be uniquely positioned to provide insight into both the evolution of DNA repair and the relations between DNA damage, DNA repair, and latent tumorigenesis.

Concluding Remarks

Based on the abundance of genetic differences among inbred strains of Xiphophorus species and the fact that interspecies hybrids are fertile, classical genetic analysis of Xiphophorus backcross hybrids has revealed linkage of genetic markers with phenotypic traits that includes hyperplastic pigment cell proliferation and tumor formation. More than two decades before the isolation of the Xmrk gene (Wittbrodt et al. 1989; Zechel et al. 1988) and the more recent isolation of the CDKN2X gene (Kazianis et al. 1999; Nairn et al. 1996b), pioneering investigator Dr. Fritz Anders had documented the hypothetical existence of both loci (Anders 1967). With a multitude of tumor models, proven tumor inducibility resulting from physical or chemical treatment regimens, and differing genetic causality leading to phenotypically identical neoplasia, the small aquarium fishes of the genus Xiphophorus offer the scientific community a valuable tool. Undoubtedly, these genetic models will be further exploited by researchers and should contribute toward the general understanding of neoplasia.

Acknowledgments

Preparation of this manuscript was supported by grants RR12253, from the National Center for Research Resources, and CA75137, from the National Cancer Institute. We appreciate the assistance of our collaborators, including Drs. Irma Gimenez-Conti, Dennis Johnston, David Mitchell, Donald Morizot, Rodney Nairn, Manfred Schartl, Richard Setlow, Juergen Vielkind, and Avril Woodhead. We also thank the staff of the Xiphophorus Genetic Stock Center for their work in producing many of the fish crosses described herein and for maintaining the 60+ pedigreed lines for the scientific community's use.

¹Abbreviations used in this article: Af, anal fin spot; BC₁, first generation backcross hybrids; BER, base excision repair; CDKN2, cyclin-dependent kinase inhibitor-2 (with CDKN2X specifically coding for a CDKN2 gene of Xiphophorus); CPD, cyclobutane pyrimidine dimmer; F₁, first filial generation; HER-1, human epidermal growth factor receptor 1; LG, linkage group; MNU, N-methyl-N-nitrosourea; O6MGMT, 0⁶ methylguanine-DNA-methyltransferase; (6-4)PD, 6-4 ultraviolet light photoproduct; PI3-kinase, phosphotidylinositol-3 kinase; RG₁, repression gene 1; Sp, spotted dorsal; UV, ultraviolet light; Xmrk-1 and -2, Xiphophorus melanoma receptor tyrosine kinase-1 and -2.

References

Adam D, Dimitrijevic N, Schartl M. 1993. Tumor suppression in Xiphophorus by an accidentally acquired promoter. Science 259:816-819.

Adam D, Maueler W, Schartl M. 1991. Transcriptional activation of the melanoma inducing Xmrk oncogene in Xiphophorus. Oncogene 6:73-80.

Ahuja MR, Anders F. 1976. A genetic concept of the origin of cancer, based in part upon studies of neoplasms in fishes. Prog Exp Tumor Res 20:380-397.

Ahuja MR, Lepper K, Anders F. 1979. Sex chromosome aberrations involving loss and translocation of tumor-inducing loci in Xiphophorus. Experientia 35:28-30.

Ahuja MR, Schwab M, Anders F. 1980. Linkage between a regulatory locus for melanoma cell differentiation and an esterase locus in Xiphophorus. J Hered 71:403-407.

Altschmied J, Baudler M, Duschl J, Schartl M. 1995. Transcriptional control of the tumor inducing gene Xmrk of Xiphophorus. (Abstract). J Cell Biochem 19A:47.

Altschmied J, Ditzel L, Schartl M. 1997. Hypomethylation of the Xmrk oncogene promoter in melanoma cells of Xiphophorus. Biol Chem 378:1457-1466.

Anders F. 1967. Tumour formation in platyfish-swordtail hybrids as a problem of gene regulation. Experientia 23:1-10.

Anders F. 1991. Contributions of the Gordon-Kosswig melanoma system to the present concept of neoplasia. Pigment Cell Res 3:7-29.

Anders A, Anders F, Klinke K. 1973a. Regulation of gene expression in the Gordon-Kosswig melanoma system I. The distribution of the controlling genes in the genome of the xiphophorin fish, Platypoecilus maculatus and Platypoecilus variatus. In: Schroder JH, ed. Genetics and Mutagenesis of Fish. New York: Springer-Verlag. p 33-52.

Anders A, Anders F, Klinke K. 1973b. Regulation of gene expression in the Gordon-Kosswig melanoma system II. The arrangement of chromatophore determining loci and regulating elements in the sex chromosomes of Xiphophorin fish, Platypoecilus maculatus and Platypoecilus variatus. In: Schroder JH, ed. Genetics and Mutagenesis of Fish. New York: Springer-Verlag. p 53-63.

Baudler M, Duschl J, Winkler C, Schartl M, Altschmied J. 1997. Activation of transcription of the melanoma inducing Xmrk oncogene by a GC box element. J Biol Chem 272:131-137.

Beaugrand JP, Goulet C. 2000. Distinguishing kinds of prior dominance and subordination experiences in males of green swordtail fish (Xiphophorus helleri). Behav Proc 50:131-142.

Borowsky R. 1973. Melanomas in Xiphophorus variatus (Pisces, Poeciliidae) in the absence of hybridization. Experientia 29:1431-1433.

Chen TT, Sarmasik A, Jang I-K, Chun CZ, Lu JK. 2001. Production of transgenic live-bearing fish and crustaceans with pantropic replication-defective retroviral vectors. Mar Biotech (in Press).

Chin L, Merlino G, DePinho RA. 1998. Malignant melanoma: Modern black plague and genetic black box. Genes Dev 12:3467-3481.

Chin L, Pomerantz J, Polsky D, Jacobson M, Cohen C, Cordon-Cardo C, Horner JWI, DePinho RA. 1997. Cooperative effects of INK4a and ras in melanoma susceptibility in vivo. Genes Dev 11:2822-2834.

Clark E. 1950. A method for artificial insemination in viviparous fishes. Science 112:722-723.

de Gruijl FR. 1999. Skin cancer and solar UV radiation. Eur J Cancer 35:2003-2009.

De Wolf W, Seinen W, Hermens JLM. 1993. N-acetyltransferase activity in rainbow trout liver and in vitro biotransformation of chlorinated anilines and benzenes in fish. Xenobiotica 23:1045-1056.

deGruiji F, Van der Leun J. 1993. Influence of ozone depletion on the incidence of skin cancer: Quantitative prediction. In: Young A, Bjorn L, Moan J, Nultsch W, eds. Environmental UV Photobiology. New York: Plenum Press. p 89-112.

Della Coletta L, Rolig RL, Fossey S, Morizot DC, Nairn RS, Walter RB. 1995. Characterization of the Xiphophorus fish (Teleostei: Poeciliidae) ERCC2/XPD locus. Genomics 26:70-76.

Dimitrijevic N, Winkler C, Wellbrock C, Gomez A, Duschl J, Altschmied J, Schartl M. 1998. Activation of the Xmrk proto-oncogene of Xiphophorus by overexpression and mutational alterations. Oncogene 16:1681-1690.

Dove AD. 2000. Richness patterns in the parasite communities of exotic poeciliid fishes. Parasitology 120:609-623.

Drexler HG. 1998. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leukemia 12:845-859.

Drobetsky EA, Turcotte J, Chateauneuf A. 1995. A role for ultraviolet A in solar mutagenesis. Proc Natl Acad Sci U S A 92:2350-2354.

Felding-Habermann B, Anders A, Dippold WG, Stallcup WB, Wiegandt H. 1988. Melanoma-associated gangliosides in the fish genus Xiphophorus. Cancer Res 48:3454-3460.

Fornzler D, Altschmied J, Nanda I, Kolb R, Baudler M, Schmid M, Schartl M. 1996. The Xmrk oncogene promoter is derived from a novel amplified locus of unusual organization. Genome Res 6:102-113.

Fornzler D, Wittbrodt J, Schartl M. 1991. Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences in Xiphophorus. Biochem Genet 29:509-524.

Frei JV, Lawley PD. 1980. Thymomas induced by simple alkylating agents in C57BL/Cbi mice: Kinetics of the dose response. J Natl Cancer Inst 64:845-856.

Friedberg EC, Walker GC, Siede W. 1995. DNA Repair and Mutagenesis. Washington DC.: ASM Press.

Goldstein AM, Fraser MC, Wallace HCJ, Tucker MA. 1994. Age at diagnosis and transmission of invasive melanoma in 23 families with cutaneous malignant melanoma/dysplastic nevi. J Natl Cancer Inst 86:1385-1390.

Gomez A, Wellbrock C, Gutbrod H, Dimitrijevic N, Schartl M. 2001. Ligand independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain. J Biol Chem 276:3333-3340.

Gordon M. 1927. The genetics of a viviparous top-minnow Platypoecilus: The inheritance of two kinds of melanophores. Genetics 12:253-283.

Gordon M. 1931. Hereditary basis of melanosis in hybrid fishes. Am J Cancer 15:1495-1523.

Gordon M. 1959. The melanoma cell as an incompletely differentiated pigment cell. In: Gordon M, ed. Pigment Cell Biology. New York: Academic Press. p 215-236.

Gutbrod H, Schartl M. 1999. Intragenic sex-chromosomal crossovers of Xmrk oncogene alleles affect pigment pattern formation and the severity of melanoma in Xiphophorus. Genetics 151:773-783.

Hannig G, Ottilie S, Schartl M. 1991. Conservation of structure and expression of the c-yes and fyn genes in lower vertebrates. Ongogene 6:361-369.

Haussler G. 1928. Uber Melanobildung bei Bastarden von Xiphophorus helleri und Platypoecilus maculatus var. Rubra. Klin Wochenschrift 7:1561-1562.

Herman JG, Jen J, Merlo A, Baylin SB. 1996. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B1. Cancer Res 56:722-727.

Herman JG, Merlo A, Mao L, Lapidus RG, Issa J-PJ, Davidson NE. 1995. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 55:4525-4530.

Hoefler CD, Morris MR. 1999. A technique for the temporary application and augmentation of pigment patterns in fish. Ethology 105:105.

Humm DG, Young RS. 1956. The embryological origin of pigment cells in platyfish-swordtail hybrids. Zoologica 41:1-10.

Ishikawa T, Sakakibara K, Kurumado K, Shimada H, Yamaguchi K. 1975. Morphologic and microspectrophotometric studies on spontaneous melanomas in Xiphophorus helleri. J Natl Cancer Inst 54:1373-1378.

Jiang P, Stone S, Wagner R, Wang S, Dayananth P, Kozak CA, Wold B, Kamb A. 1995. Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2). J Mol Evol 41:795-802.

Kallman KD. 1971. Inheritance of melanophore patterns and sex determination in the montezuma swordtail, Xiphophorus montezumae cortezi rosen. Zoologica 56:77-94.

Kallman KD. 1975. The platyfish, Xiphophorus maculatus. In: King RC, ed. Handbook of Genetics. New York: Plenum. p 81-132.

Kallman KD, Atz JW. 1966. Gene and chromosome homology in fishes of the genus Xiphophorus. Zoologica 51:107-135.

Kamb A, Shattuck-Eidens D, Eeles R, Liu Q, Gruis NA, Ding W, Hussey C, Tran T, Miki Y, Weaver-Feldhaus J, McClure M, Aitken JF, Anderson DE, Bergman W, Frants R, Goldgar DE, Green A, MacLennan R, Martin NG, Meyer LJ, Youl P, Zone JJ, Skolnick MH, Cannon-Albright LA. 1994. Analysis of the p16 gene (CDKN2) as a candidate for the chromosome 9p melanoma susceptibility locus. Nat Genet 8:22-26.

Kazianis S, Coletta LD, Morizot DC, Johnston DA, Osterndorff EA, Nairn RS. 2000. Overexpression of a fish CDKN2 gene in a hereditary melanoma model. Carcinogenesis 21:599-605.

Kazianis S, Gan L, Della Coletta L, Santi B, Morizot D, Nairn R. 1998a. Cloning and comparative sequence analysis of TP53 in Xiphophorus fish hybrid melanoma models. Gene 212:31-38.

Kazianis S, Gimenez-Conti I, Trono D, Pedroza A, Chovanec LB, Morizot DC, Nairn RS, Walter RB. 2001a. Genetic analysis of MNU-induced neoplasia within Xiphophorus hybrid fish. Mar Biotech (in Press).

Kazianis S, Gimenez-Conti IB, Setlow RB, Woodhead AD, Harshbarger JC, Trono D, Ledesma M, Nairn Rodney S, Walter Ronald B. 2001b. MNU induction of neoplasia in a novel fish model. Lab Invest (in Press).

Kazianis S, Gutbrod H, Nairn RS, McEntire BB, Della Coletta L, Walter RB, Borowsky RL, Woodhead AD, Setlow RB, Schartl M, Morizot DC. 1998b. Localization of a CDKN2 gene in linkage group V of Xiphophorus fishes defines it as a candidate for the DIFF tumor suppressor. Genes Chromosom Cancer 22:210-220.

Kazianis S, Morizot DC, Coletta LD, Johnston DA, Woolcock B, Vielkind JR, Nairn RS. 1999. Comparative structure and characterization of a CDKN2 gene in a Xiphophorus fish melanoma model. Oncogene 18:5088-5099.

Kazianis S, Morizot DC, McEntire BB, Nairn RS, Borowsky RL. 1996. Genetic mapping in Xiphophorus hybrid fish: Assignment of 43 AP-PCR/RAPD and isozyme markers to multipoint linkage groups. Genome Res 6:280-289.

Koh HK. 1991. Cutaneous melanoma. Med Prog 325:171-182.

Kosswig C. 1927. Uber Bastarde der Teleostier Platypoecilus und Xiphophorus. Zeitschrift fur induktive Abstammungs- und Vererbungslehre 44:253.

Kosswig C. 1928. Uber Kreuzungen Zwischen den Teleostiern Xiphophorus Helleri und Platypoecilus maculatus. Zeitschrift fur induktive. Abstammungs- und Vererbungslehre 47:150-158.

Kraehn GM, Schartl M, Peter RU. 1995. Human malignant melanoma: A genetic disease? Cancer 75:1228-1237.

Langley RG, Sober AJ. 1997. A clinical review of the evidence for the role of ultraviolet radiation in the etiology of cutaneous melanoma. Cancer Invest 15:561-567.

Laporte M. 1998. Clinical characteristics of malignant melanoma. Acta Chir Belg 98:139-140.

Luh FY, Archer SJ, Domaille PJ, Smith BO, Owen D, Brotherton DH, Raine AR, Xu X, Brizuela L, Brenner SL, Laue ED. 1997. Structure of the cyclin-dependent kinase inhibitor p19Ink4d. Nature 389:999-1003.

Malumbres M, Perez de Castro I, Santos J, Fernandez Piqueras J, Pellicer A. 1999. Hypermethylation of the cell cycle inhibitor p15INK4b 3'-untranslated region interferes with its transcriptional regulation in primary lymphomas. Oncogene 18:385-396.

McConnell TJ, Godwin UB, Norton SF, Nairn RS, Kazianis S, Morizot DC. 1998. Identification and mapping of two divergent, unlinked major histocompatibility complex class II B genes in Xiphophorus fishes. Genetics 149:1921-1934.

Meadow JA, Walter RB, Mitchell DL. 2000. Induction, distribution and repair of UV photodamage in the platyfish, Xiphophorus signum. Photochem Photobiol 72:260-266.

Meyer A. 1997. The evolution of sexually selected traits in male swordtail fishes (Xiphophorus: Poeciliidae). Heredity 79:329-337.

Meyer A, Morrissey JM, Schartl M. 1994. Recurrent origin of a sexually selected trait in Xiphophorus fishes inferred from a molecular phylogeny. Nature 368:539-542.

Miller BA, Gloeckler-Ries LA, Hankey BF. 1993. Cancer Statistics Review: 1973-1990. Bethesda: NIH.

Mitchell DL. 1996. Radioimmunoassay of DNA damaged by ultraviolet light. In: Pfeifer G, ed. Technologies for Detection of DNA Damage and Mutations. New York: Plenum Publishing Corp. p 73-85.

Mitchell DL, Meador JA, Byrom M, Walter RB. 2001. Resolution of UV-induced DNA damage in Xiphophorus fishes. Mar Biotech (in Press).

Mitchell DL, Scoggins JT, Morizot DC. 1993. DNA repair in the variable platyfish (Xiphophorus variatus) irradiated in vivo with ultraviolet B light. Photochem Photobiol 58:455-459.

Morizot DC, McEntire Brenda B, Della Coletta L, Kazianis S, Schartl M, Nairn RS. 1998a. Mapping of tyrosine kinase gene family members in a Xiphophorus melanoma model. Mol Carcinog 22:150-157.

Morizot DC, Nairn RS, Walter RB, Kazianis S. 1998b. Linkage map of Xiphophorus fishes. ILAR J 39:237-248.

Morizot DC, Siciliano MJ. 1983. Linkage group V of platyfishes and swordtails of the genus Xiphophorus (Poeciliidae): Linkage of loci for malate dehydrogenase-2 and esterase-1 and esterase-4 with a gene controlling the severity of hybrid melanomas. J Natl Cancer Inst 71:809-813.

Nairn RS, Della Coletta L, McEntire BB, Walter RB, Morizot DC. 1996a. Assignment of the TP53 orthologue to a new linkage group (LG XIV) in fish of the genus Xiphophorus (Teleostei: Poeciliidae). Cancer Genet Cytogenet 88:82.

Nairn RS, Kazianis S, Della Coletta L, Trono D, Butler AB, Walter RB, Morizot DC. 2001. Genetic analysis of susceptibility to spontaneous and UV carcinogenesis in Xiphophorus hybrid fish. Mar Biotech (in Press).

Nairn RS, Kazianis S, McEntire BB, Della Coletta L, Walter RB, Morizot DC. 1996b. A CDKN2-like polymorphism in Xiphophorus LG V is associated with UV-B-induced melanoma formation in platyfish-swordtail hybrids. Proc Natl Acad Sci U S A: 93:13042-13047.

Nairn RS, Morizot DC, Kazianis S, Woodhead AD, Setlow RB. 1996c. Nonmammalian models for sunlight carcinogenesis: Genetic analysis of melanoma formation in Xiphophorus hybrid fish. Photochem Photobiol 64:440-448.

Nanda I, Volff JN, Weis S, Korting C, Froschauer A, Schmid M, Schartl M. 2000. Amplification of a long terminal repeat-like element on the Y chromosome of the platyfish, Xiphophorus maculatus. Chromosoma 109:173-180.

Nanda I, Weis S, Fornzler D, Altschmied J, Schartl M, Schmid M. 1996. Clustered organization and conservation of the Xiphophorus maculatus D locus, which includes two distinct gene sequences. Chromosoma 105:242-249.

Ohshima M, Ward JM. 1984. Promotion of N-methyl-N-nitrosourea-induced thyroid tumors by iodine deficiency in F344/NCr rats. J Natl Cancer Inst 73:289-296.

Pollock PM, Pearson JV, Hayward NK. 1996. Compilation of somatic mutations of the CDKN2 gene in human cancers: Non-random distribution of base substitutions. Genes Chromosom Cancer 15:77-88.

Rauchenberger M, Kallman KD, Morizot DC. 1990. Monophyly and geography of the Rio Panuco basin swordtails (genus Xiphophorus) with descriptions of four new species. Novitates 2975:1-41.

Reed ND, Manning DD. 1978. Present status of xenotransplantation of non-malignant tissue to the nude mouse. In: Fogh J, Giovanella BC, eds. The Nude Mouse in Experimental and Clinical Research. New York: Academic Press. p 167-185.

Richie ER, Angel JM, Rinchik EM. 1996. Tlag1, a novel murine tumor susceptibility gene that regulates MNU- induced thymic lymphoma development. Prog Clin Biol Res 395:23-32.

Rigel DS, Kopf AW, Friedman RJ. 1987. The rate of malignant melanoma in the United States: Are we making an impact? J Am Acad Dermatol 17:1050-1053.

Rosenthal GG, Evans CS. 1998. Female preference for swords in Xiphophorus helleri reflects a bias for large apparent size. Proc Natl Acad Sci U S A 95:4431-4436.

Ruas M, Peters G. 1998. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim Biophys Acta 1378:F115-F177.

Sauter ER, Herlyn M. 1998. Molecular biology of human melanoma development and progression. Mol Carcinog 23:132-143.

Schartl M. 1990. Homology of melanoma-inducing loci in the genus Xiphophorus. Genetics 126:1083-1091.

Schartl M. 1995. Platyfish and swordtails: A genetic system for the analysis of molecular mechanisms in tumor formation. Trends Genet 11:185-189.

Schartl M, Hornung U, Gutbrod H, Volff JN, Wittbrodt J. 1999. Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk- oncogene deletion and gene disruption by a transposable element. Genetics 153:1385-1394.

Schartl A, Malitschek B, Kazianis S, Borowsky R, Schartl M. 1995. Spontaneous melanoma formation in nonhybrid Xiphophorus. Cancer Res 55:51.

Schartl M, Peter RU. 1988. Progressive growth of fish tumors after transplantation into thymus- aplastic (nu/nu) mice. Cancer Res 48:741-744.

Schmahl G, Schmidt H, Ritter G. 1996. The control of ichthyophthiriasis by a medicated food containing quinine: Efficacy tests and ultrastructure investigations. Parasitol Res 82:697-705.

Schuh R, Aicher W, Gaul U, Cote S, Preiss A, Maier D, Seifert E, Nauber U, Schroder C, Kemler R. 1986. A conserved family of nuclear proteins containing structural elements of the finger protein encoded by Kruppel, a Drosophila segmentation gene. Cell 47:1025-1032.

Schwab M. 1999. From fish to FISH: The comparative pathology of neuroblastomas in humans, mice and fish. J Cancer Res Clin Oncol 125:123-124.

Schwab M, Abdo S, Ahuja MR, Kollinger G, Anders A, Anders F, Frese K. 1978a. Genetics of susceptibility in the platyfish/swordtail tumor system to develop fibrosarcoma and rhabdomyosarcoma following treatment with N-methyl-N-nitrosourea (MNU). Cancer Res Clin Oncol 91:301-315.

Schwab M, Haas S, Ahuja MR, Kollinger G, Anders A, Anders F. 1978b. Genetic basis of susceptibility for development of neoplasms following treatment with N-methyl-N-nitrosourea (MNU) or x-rays in the platyfish/swordtail system. Experientia 34:780-782.

Schwab M, Kollinger G, Haas J, Ahuja MR, Abdo S, Anders A, Anders F. 1979. Genetic basis of susceptibility for neuroblastoma following treatment with N-methyl-N-nitrosourea and x-rays in Xiphophorus. Cancer Res 39:519-526.

Setlow RB. 1974. The wavelengths in sunlight effective in producing skin cancer: A theoretical analysis. Proc Natl Acad Sci U S A 71:3363-3366.

Setlow RB, Grist E, Thompson K, Woodhead AD. 1993. Wavelengths effective in induction of malignant melanoma. Proc Natl Acad Sci U S A 90:6666-6670.

Setlow RB, Woodhead AD. 1994. Temporal changes in the incidence of malignant melanoma: Explanation from action spectra. Mutat Res 307:301.

Setlow RB, Woodhead AD, Grist E. 1989. Animal model for ultraviolet radiation-induced melanoma: Platyfish-swordtail hybrid. Proc Natl Acad Sci U S A 86:8922-8926.

Siciliano MJ, Morizot DC, Wright DA. 1976. Factors responsible for platyfish-swordtail hybrid melanomas---Many or few? In: Riley V, ed. Pigment Cell. Basel: Karger. p 47-58.

Sobel HJ, Marquet E, Kallman KD, Corley GJ. 1975. Melanomas in platy/swordtail hybrids. In: Ribelin WE, Migaki G, eds. The Pathology of Fishes. Madison: University of Wisconsin Press. p 945-981.

Trainor BC, Basolo AL. 2000. An evaluation of video playback using Xiphophorus helleri. Anim Behav 59:83-89.

Venkataramani R, Swaminathan K, Marmorstein R. 1998. Crystal structure of the CDK4/6 inhibitory protein p18INK4c provides insights into ankyrin-like repeat structure/function and tumor-derived p16INK4 mutations. Nat Struct Biol 5:74-81.

Vielkind U. 1976. Genetic control of cell differentiation in platyfish-swordtail melanomas. J Exp Zool 196:197-204.

Vielkind JR, Kallman KD, Morizot DC. 1989. Genetics of melanomas in Xiphophorus fishes. J Aquat Anim Health 1:69-77.

Vielkind U, Vielkind J. 1970. Nuclear pockets and projections in fish melanoma. Nature 226:655-656.

Vielkind J, Vielkind U, Anders F. 1971. Electron microscopic studies of melanotic and amelanotic melanomas in Xiphophorin fish. Z Krebsforsch 75:243-245.

Volff J-N, Froschauer A, Koerting C, Bernhardt W, Schartl M. 2001. Genomic plasticity of the Xmrk oncogene region in the melanoma fish model Xiphophorus. Mar Biotech (in Press).

Walter RB, Rolig RL, Kozak KA, McEntire B, Morizot DC, Nairn RS. 1993. Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene. Mol Biol Evol 10:1227-1238.

Walter RB, Sung H-M, Intano GW, Walter CA. 2001a. Charcterization of O⁶-methylguanine-DNA-methyltransferase (O⁶-MGMT) activity in Xiphophorus fishes. Mutat Res (in press).

Walter RB, Sung H-M, Mitchell DL, Intano GW, Walter CA. 2001b. Relative base excision repair activity in Xiphophorus fish tissue extracts. Mar Biotech (in Press).

Weis S, Schartl M. 1998. The macromelanophore locus and the melanoma oncogene Xmrk are separate genetic entities in the genome of Xiphophorus. Genetics 149:1909-1920.

Welch DR, Goldberg SF. 1997. Molecular mechanisms controlling human melanoma progression and metastasis. Pathobiology 65:311-330.

Wellbrock C, Fischer P, Schartl M. 1998a. Receptor tyrosine kinase Xmrk mediates proliferation in Xiphophorus melanoma cells. Int J Cancer 76:437-442.

Wellbrock C, Fischer P, Schartl M. 1999. PI3-kinase is involved in mitogenic signaling by the oncogenic receptor tyrosine kinase Xiphophorus melanoma receptor kinase in fish melanoma. Exp Cell Res 251:340-349.

Wellbrock C, Geissinger E, Gomez A, Fischer P, Friedrich K, Schartl M. 1998b. Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma. Oncogene 16:3047-3056.

Wellbrock C, Lammers R, Ullrich A, Schartl M. 1995. Association between the melanoma-inducing receptor tyrosine kinase Xmrk and src family tyrosine kinases in Xiphophorus. Oncogene 10:2135-2143.

Wellbrock C, Schartl M. 1999. Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59fyn, GRB2 and Shc. Eur J Biochem 260:275-283.

Wikonkal NM, Brash DE. 1999. Ultraviolet radiation induced signature mutations in photocarcinogenesis. J Invest Dermatol Symp Proc 4:6-10.

Winkler C, Wittbrodt J, Lammers R, Ullrich A, Schartl M. 1994. Ligand-dependent tumor induction in medakafish embryos by a Xmrk receptor tyrosine kinase transgene. Oncogene 9:1517-1525.

Wittbrodt J, Adam D, Malitschek B, Maueler W, Raulf F, Telling A, Robertson SM, Schartl M. 1989. Novel putative receptor tyrosine kinase encoded by the melanoma- inducing Tu locus in Xiphophorus. Nature 341:415-421.

Woolcock BW, Schmidt BM, Kallman KD, Vielkind JR. 1994. Differences in transcription and promoters of Xmrk-1 and Xmrk-2 genes suggest a role for Xmrk-2 in pigment pattern development in the platyfish, Xiphophorus maculatus. Cell Growth Diff 5:575-583.

Yanar M, Ozbilgin MK, Polat S, Tunali N. 1996. Diagnosis of melanoma in the tuxedo swordtail (Xiphophorus helleri ´ X. maculatus, Poecillidae). Turkish J Zool 20(Suppl):309-313.

Zander CD. 1969. Uber die enstehung und veranderung von farbmustern in der gattung Xiphophorus (Pisces). Mitt Hamburg Zool Mus Inst 66:241-271.

Zarbl H, Sukumar S, Arthur AV, Martin-Zanca D, Barbacid M. 1985. Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature 315:382-385.

Zechel C, Schleenbecker U, Anders A, Anders F. 1988. v-erbB related sequences in Xiphophorus that map to melanoma determining Mendelian loci and overexpress in a melanoma cell line. Oncogene 3:605-617.

Zechel C, Schleenbecker U, Anders A, Pfutz M, Anders F. 1989. Search for genes critical for the early and/or late events in carcinogenesis: Studies in Xiphophorus (Pisces, Teleostei). Hamatol Bluttransfus 32:366-385.

p

Walter Plate 1 (A) Gordon-Kosswig cross following the inheritance of two genomic regions: linkage group (LG) XXIV (Sd, Xmrk-2) and LG V (Diff). Loci derived from X. maculatus Jp 163 A strain, carrying Sd, are blue; and the corresponding loci from the swordtail are red. Parentheses indicate specific strains. The BC₁ hybrid (bottom left) exhibits invasive melanoma causing necrosis of the dorsal fin region, which occurs spontaneously in 25% of BC₁ hybrids simply due to the interspecies cross. (B) X. helleri ´ (X. maculatus Jp 163 B ´ X. helleri) cross used in ultraviolet light and N-methyl-N-nitrosourea tumor induction. The X. maculatus Jp 163 B strain carries Sp, which consists of macromelanophore spots on the flanks of the animal. This pattern becomes enhanced upon crossing with X. helleri but does not lead to melanoma at appreciable incidences (7%) unless BC₁ hybrid fish are treated with tumor induction protocols soon after birth (see text). Note the heavily pigmented (bottom left) and lightly pigmented (bottom, second from left) phenotypes segregating in the BC₁ hybrids. This pigmentation is enhanced melanosis, but not a melanoma.

Walter Plate 2 X. couchianus ´ (X. maculatus ´ X. couchianus) crosses used in tumor induction experiments. (A) When X. maculatus Jp 163 A, harboring Sd is crossed with X. couchianus, the pigment pattern becomes largely suppressed (note, e.g., the relative absence of Sd in the dorsal fin [bottom left] animal). In contrast, the dorsal red (Dr) erythrophore pattern becomes enhanced in the same BC₁ hybrids (note orange color of 50% of the BC₁ hybrids). The loci carried by the X. maculatus parent are indicated in blue, and +/+ in red indicates the corresponding X. couchianus alleles. (B) Same cross as panel A but using the X. maculatus Jp 163 B carrying the Sp pigment pattern. Loci indicated are as described in Figure 1, with the exception that a "c" superscript indicates the X. couchianus allele. In this case, the Sp macromelanophore pigment pattern becomes severely enhanced in Sp-inheriting BC₁ hybrids, resembling the melanotic enhancement observed for Sd in the Gordon-Kosswig cross or Sp in the cross with X. helleri (see Plate 1). Despite enhanced melanosis, BC₁ hybrids from this cross do not develop tumors at appreciable incidences unless treated shortly after birth with a tumor induction protocol (see text).

Walter Plate 3 Model cross, X. andersi ´ (X. maculatus Jp 163 B ´ X. andersi). The BC₁ hybrids exhibit severe enhancement of the Sp pigment pattern, which is very similar to the enhancement observed for the heavily pigmented hybrids shown in the X. couchianus backcross (Plate 2B). Attempts at UVB induction of melanoma in this cross have not been successful (see text). However, N-methyl-N-nitrosourea-induced tumorigenesis in this hybrid model occurs at very high incidences, and genetic analyses do not indicate association of CDKN2X inheritance with melanoma development.

Walter Plate 4 Example of Af melanosis. (A) Af pattern (arrow, left) is normally difficult to discern in normal male X. maculatus Jp 163 A fish. (B) After treatment with N-methyl-N-nitrosourea, the melanocytes comprising the Af pattern often become enhanced and grow up the gonopodium and into the ventral visceral cavity region.

Copyright © 2008. National Academy of Sciences.
All rights reserved.
500 Fifth St. N.W., Washington, D.C. 20001.
Terms of Use and Privacy Statement